Serine proteases of the chymotrypsin fold are of great interest due to detailed understanding of their enzymatic properties and their role in a number of physiological and pathological processes. We have been developing the macromolecular inhibitor ecotin to be a "fold-specific" inhibitor that is selective for members of the chymotrypsin-fold class of proteases. Inhibition of protease activity through the use of ecotin and engineered ecotins has resulted in inhibition of rat prostate differentiation and retardation of the growth of human PC-3 prostatic cancer tumors. In an effort to identify the proteases that may be involved in these processes, RT-PCR with PC-3 poly(A)+ mRNA was performed using degenerate oligonucleotide primers. These primers were designed using conserved protein sequences unique to chymotrypsin-fold serine proteases. Five proteases were identified: urokinase-type plasminogen activator, factor XII, protein C, trypsinogen IV, and a protease that we refer to as membrane-type serine protease 1 (MT-SP1). The cloning and characterization of the MT-SP1 cDNA shows that it encodes a mosaic protein that contains a transmembrane signal anchor, two CUB domains, four LDLR repeats, and a serine protease domain. Northern blotting shows broad expression of MT-SP1 in a variety of epithelial tissues with high levels of expression in the human gastrointestinal tract and the prostate. A His-tagged fusion of the MT-SP1 protease domain was expressed in E. coli, purified, and autoactivated. Ecotin and variant ecotins are subnanomolar inhibitors of the MT-SP1 activated protease domain, suggesting a role for MT-SP1 in prostate differentiation and the growth of prostatic carcinomas. The Computer Graphics Laboratory facilities have been invaluable in the cloning and characterization of this novel serine protease.